It is known that the fluorophors and phosphors, namely, the so-called phosphors in the generally term, include those of the kind whose intensity of emission is decreased by certain substances. This phenomenon is called "quenching (reaction)" and a substances which induces this quenching reaction is called a "quencher." Generally, the following formula (which is called "Stern-Volmer-equation") is established between the intensity of emission and the concentration of a quencher. EQU I.sub.o /I=1+k.sub.q. .tau..sub.o [Q] (I)
wherein I.sub.o is the intensity of emission in the substantial absence of the quencher, I is the intensity of emission when the concentration of the quencher is [Q], k.sub.q is the rate constant of the quenching reaction, and .tau..sub.o is the life of the emission in the substantial absence of the molecules of the quencher, and the product (=K) of multiplied by is the coefficient of quenching.
In this formula (I), I.sub.o, k.sub.q, and .tau..sub.o are constants which are fixed by the kinds of a phosphor and a quencher contained in the system and the kind of a substance containing the phosphor. The concentration of the quencher, therefore, can be determined by finding the value of I. Where, I.sub.o, k.sub.q, and .tau..sub.o are unknown, the practice of finding the intensity of emission when the concentration of the quencher is 0 and using the intensity as I.sub.o, finding the intensity of emission when the concentration of the quencher is at a known point and using this intensity as I, plotting the ratio of I.sub.o /I relative to the concentration of the quencher, and finding the inclination of the straight line obtained by the plotting as K (=k.sub.q..tau..sub.o) prior to the determination of the concentration of the quencher is generally in vogue. The various constants mentioned above could be found by the use of a device capable of determining the life of emission. This device, however, has not been popularized because it is intricate and expensive.
Incidentally, the method which determines the concentration of a substance by making use of this phenomenon of quenching has found practical utility in various fields. For example, a device for determining the concentration of oxygen by the utilization of the quenching reaction caused by oxygen on such a phosphor as pyrene (Japanese Patent Publication SHO 59(1984)-24,379 and Japanese Patent Laid-Open SHO 59(1984)-108,958, for example), determination of the diffusion coefficient of oxygen in a macromolecular compound by the utilization of the quenching reaction caused by oxygen on such a phosphor as triphenylene (Journal of Physical Chemistry, Vol. 69, No. 11, page 3,677, 1965), and determination of the intracellular distribution of oxygen by the utilization of the quenching reaction caused by oxygen upon pyrenebutyric acid (Biochem. Biophys. Acta., Vol. 279, page 397, 1972) have been known to the art. Though in the examples cited above, the substances themselves subjected to determination function as quenchers relative to phosphors, it is allowable to use other chemical species as quencher. For example, quenchers may be derived by causing substances subjected to determination to undergo certain chemical reactions (such as, for example, "enzymatic reaction"). The determination of concentration may be carried out by utilizing such a quencher which has been derived by a chemical reaction.
In the known methods described above, the fact that the light emanating from an excitation light source and reaching a photo-detector has a constant intensity at all times and the fact that the variation or loss caused to the light signal of emission in a light transmission system extending from a phosphor through a photo-detector is fixed constitute preconditions for the determination. When such a method is put to actual use as for a protracted service, for example, the light intensity of the excitation light source itself entails a drift. When an optical fiber is used as a light transmission system, the response obtained at all is very unstable because the intensity of the light advancing from the excitation light source to the phosphor and the light signal of emission mentioned above are conspicuously affected by variations in such physical conditions as bends in the optical fiber and fluctiations of the ambient temperature.
In other words, the symbol I in the formula (I) or a modification thereof: EQU I=I.sub.o /{1+K[Q]} (II)
always includes a variable component (hereinafter represented by "v"). The intensity of light actually detected by the photo-detector, therefore, ought to be expressed as I.times.(1+v). It follows that the intensity of emission in the absence of a quencher formed of a substance subjected to determination or a substance derived from the substance similarly includes a variable component and this variable component is detected additionally by the photo-detector. Thus, the intensity I cannot be safely treated as a fixed constant throughout the entire course of determination.
To ensure constancy of the light intensity from the excitation light source, many commercially available spectrofluorometers are configured to offset time-course changes of the intensity of excited light by optically taking out in a fixed proportion of intensity a part of the light emanating from the excitation light source (generally a xenon lamp) before the light reaches a sample chamber holding a fluorescent substance, detecting the light intensity of the separated light with an exclusive photo-detector, and feeding back the electric signal from the photo-detector to the voltage being applied to a photomultiplier serving the purpose of detecting the intensity of emission. Peterson et al., in their invention Japanese Patent Laid-Open SHO 59(1984)-500,896 [PCT/US82/01418] concerning use of a probe for determining concentration of oxygen by utilizing the phenomenon that the fluorescence from perylene dibutylate fixed by adsorption on a porous carrier, recommend use of scattered light from an excitation light source as the reference light for the fluorescence.
What is common to these techniques is the fact that they are directed to offsetting the variation of the intensity of light from the light source itself by determining the intensity of a part of the light from the excitation light source. Though these techniques can cope with the variation of the intensity of light from the light source itself, they give no consideration to other variable components of the intensity of emission and, therefore, are ineffective with respect to errors of determination originating in such variations in the determination of the concentration of a substance by the utilization of the quenching described above. The technique recommended in the invention mentioned above is usable only when the light from the excitation light source is scattered at the position containing the phosphor. It produces absolutely no effect in the configuration incapable of producing the scattering or in the configuration incapable of allowing the scattered light produced at all to be introduced into the path of light leading to the detector. By this technique, even the determination is not attained when the intensity of scattered light is very feeble.
An object of this invention, therefore, is to provide a novel method and apparatus for optical determination of the concentration of a substance. Another object of this invention is to provide a method and apparatus for optical determination of the concentration of a specific substance in a liquid or a gas, which accomplishes the determination in a simple configuration with a high accuracy. Yet another object of this invention is to provide a method and apparatus for optically determining the concentration of a specific substance in a liquid or a gas by utilizing the phenomenon that the intensity of emission from a phosphor is varied by the specific substance or by a substance derived from the specific substance, which method and apparatus attains the determination by securing stable response without being conspicuously affected by variable factors.